Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling

In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling.A series of elegant experiments have demonstrated recently that phloem samples collected from cut petioles and stems of cucurbits do not represent pure fascicular phloem sap but rather the mixed content of extrafascicular phloem (EFP), xylem, and fascicular phloem (Zhang et al., 2010, 2012). The EFP is a unique feature of Cucurbitaceae. It consists of a complex network of longitudinal perifascicular strands next to the fascicular bundles, lateral commissural strands, and entocyclic as well as ectocyclic sieve tubes (Zhang et al., 2012). In contrast to the fascicular phloem, the EFP does not build effective callose plugs and freely exudes from cut sieve tubes. Due to easy sampling and its high protein content, cucurbit exudates were frequently used for phloem biochemistry (van Bel and Gaupels, 2004; Turgeon and Oparka, 2010; Atkins et al., 2011). Recently, more than 1,100 phloem proteins were identified in a large-scale proteomic approach with pumpkin (Cucurbita maxima; Lin et al., 2009). Interestingly, 67%, 46%, and 62% of the previously identified phloem proteins from rice (Oryza sativa), rape (Brassica napus), and castor bean (Ricinus communis), respectively, were found among the EFP proteins of pumpkin, confirming functional overlap between extrafascicular and fascicular phloem of different plant species (Lin et al., 2009).Although the EFP is physically and functionally linked to the fascicular phloem, a role in assimilate transport, the major function of fascicular phloem, is still ambiguous. The presence of many defense-related proteins in cucurbit phloem exudates rather pointed toward a role of the EFP in (systemic) stress and defense responses (van Bel and Gaupels, 2004; Walz et al., 2004; Turgeon and Oparka, 2010). In this regard, it has been largely overlooked by phloem biologists that phloem exudates of cucurbits are routinely classified by ecologists as latex-like exudates involved in defense against herbivorous insects (Carroll and Hoffman, 1980; Tallamy, 1985; Konno, 2011). In fact, the EFP is similar to branched laticifer (latex-containing conduits) networks, which develop from protophloem and/or phloem initials (Hagel et al., 2008). For this reason, and for better differentiation from fascicular phloem samples, hereafter we will use the term phloem latex instead of phloem exudates.Phloem latex provides two layers of defense. It is a physical barrier for small insects, which can be trapped in large droplets of exudates from wounded veins or sticky compounds that might glue their mouth parts (Konno, 2011). In addition, it was also shown that compounds in phloem latex of squash (Cucurbita spp.) such as cucurbitacin steroids deterred beetles from feeding (Carroll and Hoffman, 1980; Tallamy, 1985). Specialist feeders of cucurbits can tolerate toxic compounds in phloem latex or even use them for their own defense. Other herbivores, such as certain species from the genus Epilachna, counteract chemical defense by trenching. They isolate a circular leaf area by cutting all tissues except for the lower epidermis, this way avoiding pressure-driven exudation within the feeding area (Carroll and Hoffman, 1980; Tallamy, 1985; Konno, 2011).Some defense responses were demonstrated to be inducible by herbivore attack both in the local as well as neighbor leaves (Carroll and Hoffman, 1980; Tallamy, 1985). In this report, we analyzed by gas chromatography/mass spectrometry (GC-MS) and stable isotope-coded protein labeling (ICPL) systemic wound responses of the EFP upon leaf wounding. Overall, wounding induced jasmonate accumulation, reprogramming of the metabolism toward increased energy status, and the regulation of proteins related to carbon metabolism, signaling, and defense. This report gives a comprehensive overview of wound-inducible changes in the metabolite and the protein composition of pumpkin phloem latex, thereby providing a framework for future in-depth studies on defense responses of both EFP as well as fascicular phloem.